Sialic acids comprise a family of about 40 derivatives of the nine-carbon sugar neuraminic acid. It is a strong organic acid with a pKa of around 2.2. The unsubstituted form, neuraminic acid, does not exist in nature. The amino group is usually acetylated to yield N-acetylneuraminic acid, the most widespread form of sialic acid, but other forms exist as well (Traving et al Cell Mol Life Sci (1998) 54, 1330-1349). Sialic acids have been found in the animal kingdom, from the echinoderms upwards to humans whereas there is no hint for their existence in lower animals of the protostomate lineage or in plants. The only known exception is the occurrence of polysialic acid in larvae of the insect Drosophila. In addition there are sialic acids in some protozoa, viruses and bacteria. Sialoglycoconjugates are present on cell surfaces as well as in intracellular membranes. In higher animals they are also important components of the serum and of mucous substances.
Sialic acids have a variety of biological functions. Due to their negative charge sialic acids are involved in binding and transport of positively charged molecules like calcium ions, as well as in attraction and repulsion phenomena between cells and molecules. Their exposed terminal position in carbohydrate chains, in addition to their size and negative charge enable them to function as a protective shield for the sub-terminal part of the molecule or the cell. They can e.g. prevent glycol-proteins from being degraded by proteases or the mucous layer of the respiratory system from bacterial infection. An interesting phenomenon is the spreading effect that is exerted on sialic acid containing molecules due to the repulsive forces acting between their negative charges. This stabilizes the correct conformation of enzyme or membrane (glyco)-proteins, and is important for the slimy character and the resulting gliding and protective function of mucous substances, such as on the surface to the eye or on mucous epithelia (Traving et al Cell Mol Life Sci (1998) 54, 1330-1349). Clearly, treatment of such sialic acid containing substances with a suitable sialidase can dramatically affect the biological properties and physical characteristics of such substances. Treatment of sialic acid containing proteins with a sialidase can make them much easier to degrade by proteases, treatment of mucous substances with sialidase could strongly reduce or eliminate their slimy characteristics. Such changes would be interesting in case such proteins need processing (e.g. proteolysis) industrial processes for e.g. protein hydrolysates.
Sialic acids take part in a variety of recognition processes between cells and molecules. Thus, the immune system can distinguish between self and non-self structures according to their sialic acid pattern. The sugar represents an antigenic determinant, for example blood group substances, and is a necessary component of receptors for many endogenous substances such as hormones and cytokines. In addition, many pathogenic agents such as toxins (e.g. cholera toxin), viruses (e.g. influenza) bacteria (e.g. Escherichia coli, Helicobacter pylori) and protozoa (e.g. Trypanosome cruzi) also bind host cells via sialic acid-containing receptors. Another important group of sialic acid recognizing molecules belong to the lectins, which are usually oligomeric glycoproteins from plants, animals and invertebrates that bind specific sugar residues. Examples are wheat germ agglutinin, Limulus polyphemus agglutinin, Sambucus nigra agglutinin and Maackia amurensis agglutinin. These lectins seem to help the plant in its defense against sialic acid containing micro-organisms or plant-eating mammals. Mammalian counterparts of the lectins include selectins and siglecs (Traving et al Cell Mol Life Sci (1998) 54, 1330-1349) and have a variety of physiological roles. Sialic acids can also assist in masking of cells and molecules. Erythrocytes are covered by a dense layer of sialic acid molecules, which is stepwise removed during the life cycle of the blood cell. The penultimate galactose residue that represent signals for degradation than become visible and the unmasked blood cells are than bound to macrophages and phagocytosed. Several other examples of such masking strategy are known. Masking can also have a detrimental effect, as can be seen from some of the tumors that are sialylated to a much higher degree than the corresponding tissues. Consequently, the masked cells are invisible to the immune defense system, and the high sialic acid contents may also play a role in the lack of inhibition of further cell growth and in spreading. The masking effect of sialic acids also helps to hide antigenic sites on parasite cells, making them invisible for the system. This is the case for microbial species like certain E coli strains and gonococci (Neisseria gonorrhoea). Treatment of such species with a sialidase would affect their possibilities to hide from the immune system.
Sialidases (neuraminidases, EC 3.2.1.18) hydrolyze the terminal, non-reducing, sialic acid linkage in glycoproteins, glycolipids, gangliosides, polysaccharides and synthetic molecules. Some sialidases, called transsialidases, are also capable to perform transfer-reactions in which they transfer the sialic acid residue from one molecule to another. Sialidases are common in animals of the deuterostomate lineage (Echinodermata through Mammalia) and also in diverse microorganisms that mostly exist as animal commensals or pathogens. Sialidases, and their sialyl substrates, appear to be absent from plants and most other metazoans. Even among bacteria, sialidase is found irregularly so that related species or even strains of one species differ in this property. Sialidases have also been found in viruses and protozoa (Traving et al Cell Mol Life Sci (1998) 54, 1330-1349). Micro-organisms containing sialidases often live in contact with higher animals as hosts, for example as parasites. Here they may have a nutritional function enabling their owners to scavenge host sialic acids to use as a carbon source. For some microbial pathogens, sialidases are believed to act as virulence factors. Yet, the role of salidases as factors in pathogenesis is controversial. On the one hand they confirm the impact of pathogenic microbial species like Clostridium perfringens. On the other hand, these enzymes are factors common in the carbohydrate catabolism of many non-pathogenic species, including higher animals. They do not, however, exert a direct toxic effect (Traving et al Cell Mol Life Sci (1998) 54, 1330-1349). Instead, their detrimental effect depends on the massive amount of enzyme that is released into the host together with other toxic factors upon induction by host sialic acids under non-physiological conditions.
The mammalian sialidases are normally approximately 40-45 kDa in size. Attempts to over-express and produce mammalian sialidases to industrially interesting amounts have not been reported. Human sialidases can be lysosomal, cytosolic or membrane bound enzymes (Achyuthan and Achyuthan (2001) Comp. Biochem. Phys. Part B, 129, 29-64). The lysosomal sialidases are glycosylated enzymes. Sialidases contain conserved motifs. The most prominent conserved motif is the so-called Asp-box, which is a stretch of amino acids of the general formula -S-X-D-X-G-X-T-W- where X represents a variable residue. This motif is found four to five times throughout all microbial sequences with the exception of viral sialidases, where it is found only once or twice or is even absent. The third Asp-box is more strongly conserved than are Asp-boxes 2 and 4. The space between two sequential Asp-boxes is also conserved between different primary structures (Traving et al Cell Mol Life Sci (1998) 54, 1330-1349). The Asp-boxes probably have a structural role and are probably not involved in catalysis. In contrast to the Asp-boxes, the FRIP-motif is located in the N-terminal part of the amino acid sequences. It encompasses the amino acids -X-R-X-P- with the arginine and praline residues absolutely conserved. The arginine is directly involved in catalysis by binding of the substrate molecule. Also important for catalytic action is a glutamic acid rich region between asp-boxes 3 and 4 as well as two further arginine residues (Traving et al Cell Mol Life Sci (1998) 54, 1330-1349)
Microbial sialidases can be classified into two groups according to their size: small proteins of around 42 kDa and large ones of 60-70 kDa. The primary structure of the large sialidases contain extra stretches of amino acids between the N-terminus and the second Asp-box as well as between the fifth Asp-box and the C-terminus. It is believed that they contribute to the broader substrate specificity of the large sialidases. Like the mammalian sialidases, the bacterial counterparts contain the F/YRIP motif and several Asp-boxes. Bacterial sialidases are often implicated in mucosal infections and virulence. Because of this, the larger bacterial sialidases are not regarded suitable for the use as processing aid in food or pharma applications. Small sialidases (same size as the mammalian sialidases) have been identified in bacteria, as indicated above. I.e. Clostridium perfringens contains a small sialidase with a size of ˜40 kDa, without the extensions common to sialidases in other bacteria. This Clostridium sialidase is however not secreted by the bacterium, and is therefore also not involved in virulence (Roggentin et al. (1995) Biol Chem Hoppe Seyler 376, 569-575). It is tempting to speculate that only the bacterial sialidases with extra extensions are involved in pathogenicity. Overexpression of bacterial sialidases in E. coli generally leads to low productivity; the small Clostridium sialidase could only be produced to 1 mg/l as intracellular protein in E. coli (Kruse et al. (1996) Protein Expr Purif. 7, 415-422).
Uchida et al (Biochimica et Biophysica Acta, vol. 350, no. 2, 1974 pp 425-431) describe the screening of microbial neuraminidases which are induced by colominic acid. Among 1000 microorganisms screened, neuramidases were obtained from Sporotrichium schenckii, Penicillium urticae and Streptomyces sp. were obtained. Penicillium urticae is not a suitable production organism for food-grade sialidase, since it is a fungus involved in food spoilage, and Sporotrichium schenckii is a pathogenic fungus. Of the bacterium Streptomyces sp the species name is not determined. The MW of the sialidases mentioned in this article is unknown. In Iwamori et al (J. Biochem. 138, pp 327-334) a bacterial Arthrobacter ureafaciens sialidases is disclosed. However this bacterial neuramidase is known to be related in HIV-1 Mediated Syncytium Formation and the Virus Binding/Entry Process (Sun et al, Virology 284, pp 26-36, 2001). There is therefore a clear need for a well-produced small, non-virulent sialidase for applications in food and pharma.
Especially the finding of a secreted fungal sialidase would be beneficial, since secreted enzymes can be easily overexpressed and purified in large quantities from a fungal culture. This would reduce the cost-price for production of a sialidase dramatically.